The long-term goal of this proposal is to delineate the mechanisms of physiological perforin gene transcription. This knowledge is lacking, but is essential for optimal design of vaccines and immunotherapy that successfully prevent or treat cancer and infection. Perforin is a pore-forming molecule that is required for the ability of natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) to kill malignant or infected cells using cytotoxic granules. Natural and experimental perforin deficiency renders humans and mice susceptible to spontaneous tumors, viral infection and a lethal inflammatory disease. The basis of perforin expression in NK cells and CTL is transcription of the perforin gene. The entire genomic territory required for physiological human perforin transcription spans 150 kb and comprises 16 DNase I hypersensitive sites (DHSs);four distal DHSs comprise its locus control region (LCR) and are required in transgenes, to drive physiological transcription. The objective of this proposal is to elucidate in mechanistic detail how these long-range domains, particularly the LCR, controls perforin. To achieve this objective, the long-range chromatin remodeling that occurs across perforin during primary CTL differentiation will be characterized (Aim 1). Transgenic mice will be generated in which the human perforin LCR can be deleted conditionally;the mouse LCR will also be targeted for conditional deletion, and an internal ribosome entry site-red fluorescent protein reporter will be inserted after the perforin translational stop to tag its mRNA and trace endogenous perforin expression in individual, vital cells. Mechanics for how the LCR functions to control transcription will be determined by combining ablation of the LCR with biochemical analysis of chromatin accessibility, histone modification, and recruitment and activity of RNA pol II, at the perforin promoter (Aim 2). Finally, the cis-acting requirement of distal non-coding sequences that are deleted from null perforin alleles, in patients with human perforin deficiency, will be investigated to continue defining the essential role of perforin transcription in health and disease (Aim 3). During immune responses, special immune cells develop to kill malignant or infected body cells using a molecule called perforin. Certain people whose immune cells cannot make perforin develop cancer quickly, and usually die as infants or children. The ability of immune cells to make perforin depends on activating its gene. In this study, we will determine how immune cells activate the perforin gene and convert inert immune cells into potent killers that can eradicate cancer. This knowledge will guide vaccine design, and methods to stimulate a patients own immune cells to kill their cancer or viral infection.